CN101974440B - Candida parapsilosis fungus and application thereof in production of pu'er tea - Google Patents
Candida parapsilosis fungus and application thereof in production of pu'er tea Download PDFInfo
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- CN101974440B CN101974440B CN2010101829485A CN201010182948A CN101974440B CN 101974440 B CN101974440 B CN 101974440B CN 2010101829485 A CN2010101829485 A CN 2010101829485A CN 201010182948 A CN201010182948 A CN 201010182948A CN 101974440 B CN101974440 B CN 101974440B
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Abstract
The invention relates to candida parapsilosis fungus and application thereof in the production of pu'er tea, and belongs to the technical field of biology. The candida parapsilosis fungus of the invention is prepared by separating and purifying a producing strain with a yeast synthetic medium and a Potato Dextrose Agar (PDA) medium; the producing strain is Candida parapsilosis Glutamate Pyruvate Transaminase (GPT)-5-11 collected in China Center for Type Culture Collection in Wuhan university in Wuhan province on May 20th 2010 with a collection number of CCTCC M 2010122. The candida parapsilosis fungus is applied to the production of the pu'er tea. The candida parapsilosis fungus has the advantages that: yeast is used in the production process of the pu'er tea according to a weight proportion of 5 percent, the Gamma-Amino Butyric Acid (GABA) content of the pu'er tea is remarkably improved, the GABA content achieves 153.32 mg/100 g, on the basis of maintaining the flavor of the conventional pu'er tea, aroma and liquor color have novel characteristics, the processing time is reduced, and the candida parapsilosis fungus plays an important role in developing novel products of pu'er tea and special pu'er tea.
Description
Technical field:
The present invention relates to a kind of Candida parapsilosis fungi and the application in Leaf of Assam Tea is produced thereof, belong to biological technical field.
Background technology:
Yeast is used for fermented juice, fermentation bread and makes the food of delicious, nutrition and serve the mankind's a lot of century.Along with the development of modern biotechnology, people utilize them in extensive diversified fermenting process, and it is more important that yeast just seems, purposes is wider.Because yeast protein content height can be used for making food-yeast or fodder yeast, its development of resources potentiality are very big.Simultaneously, the yeast thalline contains amounts of protein and abundant VITAMINs, can edible, medicinal and processing feed, and the biological activity of organism materials such as protein, VITAMINs, amino acid behind saccharomycetes to make fermentation increases substantially.Yeast has obtained to be widely used in leavened food; Has very high security; Also exist higher GAD active, can improve alpha-aminobutyric acid content in the fermented liquid, people such as Takahashi have screened GABA transaminase and succinic semialdehyde dehydrogenase defective mutant bacterial strain GAB7-1 and the GAB7-2 of Saccharomyces cerevisiae UT-1; GABA concentration has reached 0.4mM and 0.42mM respectively in its fermented liquid, has improved 2.0 and 2.1 times respectively than wild strain.
(γ-aminobutyric acid GABA), is the new resource food that a kind of nonprotein is formed to γ-An Jidingsuan, and the physiologically active that tool is extremely strong extensively is present in the natural animal and plant body.Plant is as all containing GABA in rhizome leaf, seed and the tissue juice such as ginseng genus, pulse family genus, herbal medicine, tea tree; In animal body; GABA only is present in almost that (content in the cerebral tissue is approximately 0.1～0.6mg/g) in the nervous tissue; It is inhibitory nerve mediator important in the mammalian central nervous system; Participate in multiple Metabolic activity; Have very high physiologically active, like step-down, improve brain function, hypermnesis, anxiety, treatment epilepsy, control asthma, regulate hormone secretion, prevent obesity, promote reproduction, activation liver kidney, improve neurocyte property old age stupid, alleviate physiological functions such as headache that cerebral thrombosis, cerebral arteriosclerosis causes, tinnitus.On September 27th, 2009; Ministry of Health's approval γ-An Jidingsuan, colostrum basic protein, conjugated linolic acid, conjugated linoleic acid glyceride, plant lactobacillus (bacterial strain ST-III), eucommia ulmoides seed oil are new resource food; Indicate that GABA gets into brand-new epoch at home, gets into the new food consumption epoch with guide Chinese.
Leaf of Assam Tea is the characteristic tea in Yunnan, and in its course of processing, pile-fermentation is one of critical process, and mikrobe is again the material impact factor of pile-fermentation operation, thus processing of mikrobe and Leaf of Assam Tea and Leaf of Assam Tea quality be formed with substantial connection.Foreign study shows; In the wet heap process of Leaf of Assam Tea; Yeast is one of dominant bacteria, can suppress the growth that some produce malicious mould, and yeast is not secreted deleterious antibiont; Yeast also has abundant nutrition such as VITAMINs, protein etc. simultaneously, to the significance that is formed with of Leaf of Assam Tea quality.
If quantity control is proper in the wet heap process of Leaf of Assam Tea, effective nutritive substance that can increase in the Leaf of Assam Tea reaches the material that human body is had health-care effect, helps forming quality styles such as Leaf of Assam Tea is sweet, pure, perfume (or spice).For this reason, the yeast fungus of we the product GABA that obtains from the occurring in nature separation and purification capable of using acts on the Leaf of Assam Tea production process, improves GABA content in the Leaf of Assam Tea, researches and develops new local flavor Leaf of Assam Tea.
Summary of the invention:
The objective of the invention is Leaf of Assam Tea to be studied, for manufacturing enterprise provides the GABA Leaf of Assam Tea useful bacterium source from the angle of mikrobe.Another object of the present invention is through the ratio of control probiotics in Leaf of Assam Tea is produced, for promoting that yunnan puer tea plays a role.
Candida parapsilosis fungi of the present invention; From crude substance, obtain through yeast synthetic medium, the separation and purification of PDA substratum; Qualitative through thin-layer chromatography; Performance liquid and amino acidanalyser quantitatively this bacterial strain product GABA content reach 2.42g/L, for producing the GABA functional foodstuff bacterium source are provided.The GPT-5-11 bacterial strain is deposited in Chinese typical culture collection center, and the address is Wuhan University school of life and health sciences preservation center, preservation date: on May 20th, 2010, preserving number: CCTCC M 2010122.
The morphological feature of Candida parapsilosis bacterium of the present invention (Candida parapsilosis) GPT-5-11 is:
YM culture medium culturing 72 hours, microscopic morphology was that cell is oval, oblong, and multipole budding has pseudohypha; Colonial morphology is cream-colored, smooth moistening, glossy, neat in edge.
The Meg 4.0 achievement results of Candida parapsilosis bacterium (Candida parapsilosis) GPT-5-11 are:
26S rDNA D1/D2 sequence map to the survey bacterial strain is proofreaied and correct; Submit GenBank ' to and at GenBank; EMBL; Carry out homologous sequence search (BLASTsearch) in international nucleic acid sequence data such as the DDBJ storehouse, with the similarity degree of compare test bacterial strain with the corresponding sequence of barms type strain, and combining form is learned and the physiologic character analysis is made evaluation to it.Sequence between bacterial strain is relatively accomplished with Clustal.x software.Carry out the molecular systematics analysis with the Neighbor-Joining method, and carry out the bootstrap statistical test 1000 times, set with TreeView software indicating system.
The present invention obtains after conventional processes such as yeast synthetic medium, the separation and purification of PDA substratum.
The present invention is applied to Leaf of Assam Tea and is processed with the effect that improves GABA content in the Leaf of Assam Tea.
The invention has the advantages that: the production process that yeast is used for Leaf of Assam Tea by weight the ratio that is 5%; GABA content in the Leaf of Assam Tea significantly improves; GABA content reaches 153.32mg/100g, and on the basis that keeps traditional puer tea flavor, fragrance and Tang Se aspect also have new characteristics; And shortened process period, the exploitation of Leaf of Assam Tea product innovation and characteristic Leaf of Assam Tea has been played a significant role.
Embodiment:
The present invention obtains after traditional yeast synthetic medium and PDA substratum separation and purification operation.
The purifying of embodiment 1:GPT-5-11 bacterial classification
Separation and purification from crude milk, pickles etc. is coated diluted sample on the yeast synthetic medium, cultivates 48h for 28 ℃, and picking list bacterium colony line purifying obtains the pure bacterial strain of yeast, obtains 23 strains altogether.The yeast synthetic medium is: glucose 20g, and yeast extract 5g, peptone 10g, agar powder 20g, water 1000mL, pH 6.0; 121 ℃ of sterilization 20min.
Purification process is following:
(1) with solid medium (yeast synthetic medium) fusing, be cooled to 45 ℃, inject sterile petri dish, about 15～20 milliliters of every ware, rocking-turn is a little left standstill, and makes dull and stereotyped subsequent use.
(2) dilution: be diluted to 10-8 to bacteria suspension by decimal dilution method, the bacteria suspension that ten times of methods are meant next pipe is than rare ten times of a last pipe.
(4) inoculation: dull and stereotyped 9 covers of previously prepd, label is 10-6,10-7,10-8 respectively, each extent of dilution is write three cover plates, so that relatively.Inject the corresponding bacteria suspension of 1ml in the plate of label at each, be coated with evenly, put into 25～28 ℃ of cultivation 24 h observation with spreading rod.
(5) cultivate after 2～5 days, the single bacterium colony of picking carries out the bacterial strain activation with the PDA slant medium and goes down to posterity, and per 4 weeks of bacterial strain switch through 1 time.Bacterial strain is transferred to 26 ℃ of activation culture 48h on the new PDA solid plate, is transferred to seed culture medium 24h again as ferment-seeded.
(6) arrive liquid substratum to the bacterial classification inoculation of selecting at last, 50mlCza substratum (glucose 30g, NaNO 33g, yeast extract 1g promptly pack in the 250ml triangular flask; K2HPO 1g, MgSO47H2O 0.5g, KCl 0.5g, FeSO4 7H2O 0.01g;, water 1000mL, pH 6.0; Contain 0.5% L-glutamic acid), the inoculum size by 2% inserts bacterial strain, and 28 ℃ of shaking tables are cultivated 48h, and it is qualitative through thin-layer chromatography to get fermented liquid, GABA content in performance liquid, the amino acidanalyser quantitative analysis fermented liquid, the screening of producing the GABA bacterial strain.
Embodiment 2: saccharomycetic production application
Gross tea is undertaken by traditional method damp and hot, ambient moisture 70%; Tidewater amount 30%, the inoculation weight ratio is that 5% GPT-5-11 lets its fermentation in gross tea subsequently, when heap temperature rise to 26 ± 3 ℃; GPT-5-11 begins to carry out metabolism; And, secrete a large amount of enzymes, especially L-Glutamic decarboxylase (GAD) along with the heap temperature further raises; L-glutamic acid can be converted into GABA under the effect of this enzyme, improve GABA content in the fermented tea appearance.
The Leaf of Assam Tea of behind inoculation Candida parapsilosis bacterium, producing, its GABA content is significantly improved, and the GABA content in the Leaf of Assam Tea reaches 153.32mg/100g, the GABA Leaf of Assam Tea of formation, its soup look red is dense bright, and flavour alcohol is slided back sweet, peculiar fragrance.
Claims (1)
1. a Candida parapsilosis (Candida parapsilosis) GPT-5-11, preservation date: on May 20th, 2010, preserving number CCTCC M 2010122, depositary institution: Chinese typical culture collection center, address: Wuhan. Wuhan University.
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| CN102919463A (en) * | 2012-11-12 | 2013-02-13 | 左权县桐峪镇嘉百岁农民专业合作社 | Preparation method for fructus forsythiae leaf fermented tea |
| CN103060203A (en) * | 2012-12-21 | 2013-04-24 | 楚雄师范学院 | Fungus and application thereof |
| CN104304531A (en) * | 2014-11-08 | 2015-01-28 | 云南滇泊洱生物科技有限公司 | Pu'er tea yeast fermentation technology |
| CN105754887B (en) * | 2014-12-16 | 2020-02-21 | 勐海茶业有限责任公司 | Lactobacillus pentosus for producing gamma-aminobutyric acid and application of lactobacillus pentosus in production of Pu' er tea |
| CN107232338A (en) * | 2017-06-22 | 2017-10-10 | 中国科学院昆明植物研究所 | A kind of method and its application that Pu'er tea is produced using special exogenous biological enzyme fermentation |
| CN113943663B (en) * | 2020-07-17 | 2024-04-09 | 中粮集团有限公司 | Candida stellati in black tea fungus and application thereof |
| CN114568555A (en) * | 2022-04-12 | 2022-06-03 | 贵州大学 | Processing method for improving GABA of gold bud green tea and gold bud green tea |
| CN117461691A (en) * | 2022-07-20 | 2024-01-30 | 安琪酵母股份有限公司 | Modified milk containing gamma-aminobutyric acid yeast extract and preparation method thereof |
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| CN1298839C (en) * | 2005-07-28 | 2007-02-07 | 秘鸣 | Yeast fungus for making wine and its application in production of puer tea |
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Application publication date: 20110216 Assignee: Xinfeng Jiangxi Xiang Industry Development Co., Ltd. Assignor: Yunnan Agricultural University Contract record no.: 2014360000137 Denomination of invention: Candida parapsilosis fungus and application thereof in production of pu'er tea Granted publication date: 20120530 License type: Exclusive License Record date: 20140626 |
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